Fig. 2. Differences in the expression, localization and activity of cathepsin B in AS-cav-1 HCT 116 cells. (A) 1 µl total RNA isolated from control and AS-cav-1 HCT 116 cells was subjected to reverse transcription followed by PCR using primer sequences to cathepsin B and ß₂-microglobulin (as a control). PCR products were resolved on a 1.2% agarose gel stained with ethidium bromide. (B) Conditioned media from control and AS-cav-1 HCT 116 cells were collected and the cells were solubilized in lysis buffer containing 0.1% Triton X-100. Media and cell lysates (40 µg protein) were analysed by SDS-PAGE and immunoblotted with anti-human-cathepsin-B antibodies. Immunoblots are representative of at least three experiments. (C) Conditioned media and cell lysates from control and AS-cav-1 HCT 116 cells were assayed for cathepsin-B activity. Intracellular (cell lysates) and extracellular (media) cathepsin-B activities were measured against Z-Arg-Arg-NHMec substrate and activity was expressed as pmoles min^–1 (µg DNA)^–1. The graphs are representative of at least three experiments and presented as means ± standard deviation (s.d.). **P<0.01. (D) Equal amounts of protein for control and AS-cav-1 HCT 116 cells were subjected to subcellular fractionation on a sucrose gradient after homogenization in sodium-carbonate buffer, pH 11.0. Fractions were collected from the top of the gradient and equal-volume aliquots of fractions 2-11 were analysed by SDS-PAGE and immunoblotted with anti-human-cathepsin-B antibody. Immunoblots are representative of at least three experiments.